KR100237144B1 - Herpes simplex virus-1 deletion variants and vaccines thereof - Google Patents

Abstract

Novel Herpes simplex viruses and vaccines based on such novel HSV-1 strains are described. In particular, viruses having a deletion in the terminal portion of RL are provided. The virus can be further modified to express heterologous antigens and also engineered to overproduce HSV Light particles. This is achieved by incorporating a ts mutation into the UL26 gene.

Description

Herpes simplex virus-1 deletion strain and method of making the same

The present invention relates to a variant of herpes simplex virus type 1 (HSV-1) lacking neuropathy. This variant is valuable in the manufacture of live, weakly toxic vaccines to prevent human HSV infection.

Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) are important human pathogens that infect more than 80% of the general population and cause recurrent mucosal skin lesions. After replication, HSV enters the peripheral nervous system where active replication is completed by unknown mechanisms. Thereafter a latent infection in the neuron is established, which persists for the life of the host. HSV can be reactivated from a latent state to produce infectious disorders. HSV is the cause of a broad spectrum of clinical disease ranging from relatively harmless skin disorders to fatal viral encephalitis.

A great deal of research has already been devoted to elucidating the genetic makeup of both HSV-1 and HSV-2. The HSV-1 genome is a new double stranded DNA molecule of approximately 152 kilobase pairs consisting of two components L and S. Each component consists of unique sequences U ₁ and U _S flanked by the inverted repeat. The construction of the HSV-2 genome is similar but not identical. See McGeoch, 1987 for a detailed description of the genetic makeup of HSV-1 and HSV-2.

The identification of genes involved in the elucidation of viral pathogenicity and its exact function is of fundamental importance for understanding the biology of herpes simplex virus (HSV). Numerous variants of both HSV type 1 (strain 17) and HSV type 2 (strain HG 52), with deletions defined in the unique and repeat sequences of both long and short regions of the viral genome, have already been isolated and characterized (Brown). 1984, Harland and Brown 1985, Brown and Harland 1987, MacLean and Brown, 1987a and b, Harland and Brown 1989). However, little is known about the molecular mechanisms regulating the neuropathy of HSV. The deletion strain of HSV-2 strain HG52, designated JH2604, was found to be non-toxic for inoculation of rats (Toba , 1989a). JH2604 has 1488 base pair deletions in two copies of the long repeat region of the genome (ie, the terminal long repeat (TR _L ) and the inverted long repeat (IR _L ) region).

The HSV-1 strain 17 / HSV-2 strain HG 52 recombination, designated RE6 (first isolated from the Institute of Virology, Glasgow by Marsden's group, 1978), has also been reported to be nontoxic in rats (Thompson , 1989).

In HSV-1, the inverted repeats of the L component named ab and b'a 'are approximately 9 Kbp, respectively, while the S components c'a' and ca are approximately 6.5 Kbp, respectively. The sequence shared by the inverted repeats of the L and S components is named 'a' sequence. It has been reported that this sequence contains a transcription initiation site and promoter-regulatory sequence for a diploid gene located in sequence b of the inverted repeat of the L component (Chou and Roizman 1986). Working with HSV strain F, these authors reported that there was an open reading frame (ORF) transcribed between the 'a' sequence and the immediately preceding gene named IE1. Using antipeptide sera they could reveal that ORF designates a protein named ICP 34.5 (Ackermann). 1986). Recently Chou and Roizman (1990) reported that their now predicted ORF was preserved in two other HSV-1 strains analyzed but not in Glasgow strain HSV-1 (17) syn +. Chou et al. (1990) suggested that neuronal loci of HSV-1 map with ICP 34.5 and require expression.

Surprisingly, 17 variants of the HSV-1 Glasgow strain modified at the distal end of R _L were found to lack neuropathy.

These strains cannot replicate in CNS neurons, but can replicate in peripheral tissues, leading to good immunological and cellular mediated responses in mice. This ability highlights the vaccine efficacy of these vaccines.

According to the present invention there is provided a HSV-1 strain which is a genome in which the terminal portion of R _L is modified in Bam HIS (0-0.02 and 0.81-0.83mu).

Bam hi This means that approximately 3Kb Bam HI in the HSV R _L region It can be appreciated that each copy of the segment is a copy.

The term “modified” as used herein refers to Bam HI due to the deletion of one or more nucleotides. Any other alteration, or substitution, of a nucleotide sequence, such as fragmentation, insertion or rearrangement of additional nucleotides.

HSV-1 may be a deletion strain that is isolated at the same time or may be a wild type strain into which a desired modification has been introduced.

Such modifications in HSV strains can be made by genetic engineering, such as by site-engineered mutations or by deleting portions of the genome that have not been substituted or replaced with pre-made DAN cassettes that join the necessary modifications. . Alternatively, it may be an isolate, such as a deletion variant, that naturally causes the HSV-1 variant.

Preferably the HSV-1 strain of the present invention is 17 variants of Glasgow strains.

In one preferred aspect the HSV-1 strain comprises a Bam HI between the Alu I sites of 125074np and 125972np in the sequence. At least 100 nucleotides in the region and its counterpart sequence in the TR _L are deleted.

More preferably Bam HI 0.5 to 3 kb of the region and its counterpart in TR _L are deleted and more preferably about 0.7-2.5 kb are deleted.

In one particular embodiment the HSV-1 variant is a deletion variant that coincides with variant 1702 and Bam HI of the R _L region as described below. A strain designated 1714, lacking 759 bp in each copy of the fragment, the deletion associated with non-neuropathy is located between nucleotide positions 125213 and 125972.

This deletion removes one complete copy of the 18 bp DR ₁ element of 'a' sequence and terminates the 1105 bp upstream of the 5 ′ end of Gene 1 adjacent to it.

In another specific embodiment, the HSV-1 variant is a variant named 1716, wherein the 759 bp deletion in variant 1714 was introduced into wild type Glasgow strain 17 ⁺ .

See Figure 1 below for a more clear understanding of the invention. 1 shows: (a) shows HSV-1 genome in circular orientation (in map units); And (b) Bam HI Show zoomed in. AluI (A) sites flanking the deletion side at Bam HI (B) and 1714/1716 are indicated. All coordinates are based on the numbering of McGeoch's group (1988). In addition, the open reading frame (R _L ORF) with 189bp conserved between the position of the 5 ′ end of IE1, the 'a' sequence, the DR ₁ / U _b combination in the 'a' sequence, and HSV-1 and HSV-2 End point of 759 bp deletion in 1716. The deletion extends from the DR ₁ / U _b binding to remove 5′107 bp of R _L ORF.

The present invention further provides a whole virus vaccine containing the HSV-1 strain according to the present invention, wherein the vaccine includes immunoprotective and non-toxic amounts of the strain of the present invention. The vaccine may contain the strain alone or in combination with other antigens and / or adjuvants.

Due to their non-pathogenic nature, the viruses of the present invention are unexpected candidates for further modification. For example, they may be further modified to carry heterologous antigens. Viruses can be treated to express antigens from HSV-2, such as HSV-2 gD. The virus induces CTL responses against antibodies and type 1 and type 2 viruses, and also enhances the overall immune response. Similarly, other antigens from other pathogens can be expressed with the viruses of the invention. For example, gene products and other envelope viruses from HCMV, VZV, EBV, HHV6, HHV7 and HIV may also participate.

In addition, the viruses of the invention can be modified by introducing mutations, typically temperature sensitive mutations, into the gene UL 26a encoding the capsid protein, P4O (Liu & Roizman 1991 a + b).

The mutation at unacceptable temperatures (typically 38.5 ° C.) results in overproduction of light particles; It is a viral location that lacks nucleocapsids and nucleic acids and thus lacks infectivity. J. of Gen Virology (1991) p661 Szilagyi and Cunningham.

Accordingly, the present invention provides lightweight particles derived from the virus described herein.

In additional embodiments, the present invention provides herpes virus light weight particles that carry heterologous antigens. For example, in one embodiment of the present invention, HSV-1 1716 was modified to express HSV-2 gD and also to contain a temperature sensitive mutation in the UL26a gene at 38.5 ° C .; This mutant overproduces lightweight particles containing HSV-2 gD. Other HSV-2 proteins can also be joined into the virus, especially the HSV-2 gene products ICPO, ICP4 and Vmw 65kD. There may be membrane proteins from other herpes viruses such as HCMV, VZV, EBV, HHV6, HHV7, and other enveloped viruses such as HSV-1 and HSV-2. For example, gB from HCMV, gp120 from HSV-1 or HSV 2 can be incorporated into viral light particles. In theory any heterologous membrane protein that does not interfere with the entry of the virus into the cell can be carried by the lightweight particles according to the invention.

Accordingly, the present invention provides herpes virus light weight particles that carry heterologous antigens. In particular, the present invention provides light weight particles carrying a simple herpes virus, preferably type 1, heterologous antigen. Embodiments of this aspect of the invention are HSV-1 1716, gD1 ⁺ , gD2 ⁺ , UL26a And lightweight particles therefrom.

The lightweight particles of the present invention can be produced by modification of the method of Szilagyi & Cunningham (see above). Briefly, cells are infected with 5 pfu / cell at an unacceptable temperature (npt) of 38.5 ° C. and the superior virus is harvested 30 minutes after infection. This formulation is centrifuged at 12K for 2 hours on a preformed 5-15% Ficoll (prepared in Eagle's medium) gradient. The light particle strip was removed with a 26G needle and pelleted at 20K overnight in normal cell growth medium (Eagles).

Lightweight particles of the invention are useful for vaccine applications. Thus in a further aspect of the present invention there is provided a vaccine containing a light virus from herpes virus carrying a heterologous antigen. In additional terms, BamHI Provided is a vaccine containing HSV-1 virus light weight particles derived from a virus comprising a modification at the terminal portion of R _L in (0-0.01 and 0.81-0.83mu).

Alternatively, or in addition to the modification (s) mentioned above, the viruses of the invention can be modified by introducing mutations, typically deletions that render the LAT promoter ineffective. The mutation further raises the level of stability, reducing both the frequency and the rate of reactivation from the incubation period.

Therefore, the present invention is BamHI The terminal portion of R _L in (0-0.02 and 0.18-0.83mu) is modified and also provides a modified HSV-1 virus to render the LAT promoter ineffective. The modified virus can be further modified to produce a heterologous antigen such as HSV-2 gD in the contemplated manner. Additionally or alternatively to heterologous antigen expression, temperature sensitive mutations can be joined into gene UL26a to enable overproduction of lightweight particles to reduce the amount of potentially infectious viruses present. The lightweight particles can be separated from the infectious virus by Ficoll centrifugation of the virus particles. Usually, the ratio of heavy particles to light particles in the light weight particle band will be 1:10 ³ , but the ratio of heavy particles to light particles is typically about 1:10 ⁶ if mutations are added within UL26a.

The present invention also provides a method for producing a whole virus vaccine comprising mixing a suitable excipient or adjuvant with a strain according to the present invention.

For the preparation of live weak vaccines, standard methodologies can be used.

In a further aspect, the present invention provides a method of treating HSV infection in a human, comprising administering an immunologically effective amount of a vaccine according to the invention to a human subject in need thereof.

The method of administering the vaccine of the present invention may be any suitable route for delivering an immunoprotective amount of a strain or lightweight particle of the present invention to a subject. However, the vaccine is preferably administered parenterally via the intramuscular or subcutaneous route. Other methods of administration can also be used, if desired, as oral administration or in other parenteral routes, ie intradermal, intranasal, or intravenous.

Appropriate immune protective and non-toxic doses of the vaccine can be readily determined by one skilled in the art. That is, the appropriate immunoprotective and non-toxic amounts of the strains or lightweight particles of the invention contained in the vaccine of the invention are within the range of an effective amount of the antigen in a conventional whole virus vaccine. However, specific dosage levels for any particular patient may include age, overall health, gender, and patient's diet; Dosing time; Route of administration; Synergistic effect with any other drug administered; And the degree of protection desired. Of course, administration can be repeated at appropriate intervals if necessary.

The following examples illustrate the invention.

[Way]

[cell]

Hamster Kidney Clone 13 cells (BHK21 / C13) (MacPherson and Stoker 1963) were cultured at twice the normal concentration of vitamins and amino acids, 5% (v / v) tryptose phosphate broth and 10% (v / v) calf serum (ETC). Proliferation was carried out in Eagle's medium containing 10).

[Virus]

Grow the virus buildup and as previously described (Brown 1973) in BHK21 / C13 cells. The parental HSV-1 strain was Glasgow strain 17 (Brown). 1973). Variant 1702, which lacks four normally occurring HSV-1 Xbal sites, is a Mobilis with 1714 isolated (MacLean and Brown 1987a).

[Restriction Enzyme Analysis of Virus Genome]

Restriction enzyme analysis was performed by a modification of Lonsdale's technique (1979). BHK21 / C13 cells were infected in the presence of ³² Pi in Eagle's medium containing 1% (v / v) calf serum and incubated at 31 ° C. for 48 hours. Virus DNA was extracted with SDS and precipitated phenol and ethanol. DNA was treated with various restriction enzymes using manufacturer's recommendations. Digestions were analyzed by electrophoresis on agarose gels at appropriate concentrations (0.5-0.8%) in TBE buffer (89 mM-tris, 89 mM boric acid, 2 mM sodium EDTA). The gel was air dried and exposed to Kodak XS1 film.

[DNA-DNA Hybridization]

DNA fragments from restriction endonuclease digests were transferred from agarose gels to a high bond nylon membrane (Amersham) by Southern method (1975) and cloned into PAT153. Hybridization with randomly disclosed DNA prepared from fragments.

Hybridization was performed in hybridization buffer containing 7% SDS and 0.5M NaP, pH 7, at 65 ° C. for 16 hours. No prehybridization was performed. Filter as described earlier (Brown 1984).

[Animal inoculation]

Three week old BALB / C mice (Bantin and Kingman) were inoculated intracardially as individual virus accumulation. Mice were anesthetized with ether and inoculated with 0.025 ml of appropriate virus diluent in phosphate buffered silane (PBS) 5% calf serum into the central region of the left hemisphere. Four mice were inoculated with viruses at doses of 10 ¹ to 10 ⁷ pfu / animal, respectively. The virus accumulation was always retired on the day of inoculation to determine the correct titration of inoculation. Mice were observed daily after inoculation and LD ₅₀ was calculated by the formulas of Reed and Muench (1938) based on deaths up to 21 days. Brains were removed from the dead animals after inoculation, homogenized, sonicated and the resulting suspension titrated against BHK21 / C13 cells. Virus plaques were picked and their restriction enzyme profiles were measured as follows.

[Virus Growth Characteristics of Living Organisms]

BHK21 / C13 (2 × 10 ⁶ ) cells were infected with a moi of 5 pfu / cell. Adsorption was performed at 37 ° C. for 45 minutes, washed twice with phosphate buffered saline containing 5% CS and continued incubation at 37 ° C. after addition of 2 ml overlay of Eagle's medium containing 10% CS. Samples were harvested at 0, 2, 4, 6, 8, 12, 16 and 24 hours and the virus released by sonication was titrated at 37 ° C.

[Thymidine Kinase Assay]

The method used was that of Jamieson and Subak-Sharpe (1974). BHK21 / C13 cells were imitated or infected with a wild type or mutant virus with a pool of 5 pfu / cell. After 1 hour of adsorption at 37 ° C. and 6 hours of incubation at 37 ° C., the cells were scraped into cold PBS and pelleted. The pellet is resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 2 mM MgCl ₂ , 10 mM NaCl, 0.5% v / v Nonidet P40, 6.5 mM 2 mercaptoethanol) kept on ice for 5 minutes, mixed briefly and In addition, it was put back on ice for 5 minutes. The sample was centrifuged and the supernatant was kept intact. 5 μl of the extract was mixed with 50 μl of total volume of reaction buffer (0.5 M Na ₂ PO ₄ pH 6, 100 mM MgCl ₂ , 2 mM dTTP, 100 ml ATP, 5 μl aqueous Me ³ H thymidine 1 m (C / ml)).

After incubation for 1 hour the reaction was stopped by the addition of 10 μl of 100 mM EDTA and 1 mM thymidine. Samples were heated at 100 ° C. for 3 minutes and placed on ice for 3 minutes. After centrifugation, the supernatant was spotted on a DE81 disc and washed three times (10 min at 37 ° C. each time) with 4 mM ammonium formate pH 6.0 and 10 μM thymidine. After two additional washes with ethanol the discs were dried and radioactivity due to 3H thymidine was measured.

[Introduction of Deletion into Wild-type Glasgow Strain 17]

1714 cloned new BamHI to introduce 1714 deletion into wild type strain 17 Linearized with BamHI and the fragments were co-trans defect with 1, 2, 5, 10 and 20 fold molar excess with the free DNA from the 17 ^+.

The resulting individual plaques were isolated and their DNA assayed by the method of Lonsdale (1979).

The virus, indicating that it had an obtained deletion, was an additional three times purified plaque before growing the virus accumulation.

[Sequence Analysis]

1714 new BamHI Fragments were cloned into the BamHI site of pGEM 3z using standard procedures (Maniatis 1982).

Positive clones were identified by restriction enzyme analysis and confirmed by Southern blotting using whole HSV-1 DNA and randomly initiated DNA from positive clones. Additional restriction enzyme analysis confirmed that the deletion was approximately 800 bp in size and within a 2.8 kb AluI fragment. This fragment was eluted from the gel and digested with SmaI and several small fragments were subcloned to M13mp8. Single stranded template DNA was prepared and sequenced using ³⁵ s labeled dATP by the method of Sanger et al. (1980). The sequencing product was run on a single concentration 6% acrylamide, 1 × TBE, 8.3 M urea gel.

[Latent study]

Right back portions of three week BALB / C mice (Bantin & Kingman) were vaccinated as described previously (Clements & Subak-Sharpe 1983, 1988). Simultaneously with inoculation, the virus was titrated on BHK21 / C13 cells to confirm the exact amount administered. For each virus, a series of 100-fold dilutions were inoculated, mice were observed, and daily clinical symptoms were recorded. At 6 weeks after the mice survived the presence of latent viruses was observed. The mice are killed, dissected to remove the two lowest thoracic ducts, six lumbar sections and the two best sacral ganglia from the inoculation side, placed in the culture medium and transferred to a culture medium that inhibits BHK21 / C13 cells, on the second day of infection. The release was screened. Inoculated BHK21 / C13 cells were stained and incubated at 37 ° C. for 2 days before observing the presence of virus plaque or cpe.

Example 1

(a) Isolation and Genomic Analysis of Variant 1714

To study recombination in HSV, we constructed a virus that had no specific restriction enzyme sites to be used as unselected markers (Brown). ; Harland and Brown 1985; Maclean and Brown 1987c). HSV-1 strain 17 mutant 1702 (MacLean and Brown 1987c) (without four HSV-1 Xbal sites and no TK ⁻ ) was Mobilus used to remove various HindIII sites by site manipulation mutagens. DNA from virus isolate H1, derived from 1702 but lacking the 0.91 mu HindIII site, was cotransfected with a mutant plasmid with no 0.18 mu HindIII site. A large number of subsequent plaques were picked up and their DNA was subjected to restriction enzyme analysis. In addition to successfully separating the desired mutants with missing 0.18mu HindIII sites, viruses with variant RE profiles not associated with loss of HindIII sites were detected.

In KpnI digestion of 1714 DNA, KpnI (2.4 × 10 ⁶ mw) was found to be lost and about 1.9 × 10 ⁶ new bonds were found. KpnI Is the terminal end of Rr (0-0.025mu and 0.805-0.83mu) and is a linking fragment And To form. 1714 It can be seen that the fragment runs faster at the edge than its equivalent wild string fragment but does not see a change in the run at the top of the gel. Similarly HpaI (3.6 × 10 ⁶ mw) is lost and a new strip of approximately 3.1 × 10 ⁶ molar weight It was detectable driving below.

HpaI (0-0.036mu and 0.79-0.83mu) is found in connection with the fast-running than 1714 ⁺ 17 at the edge of water And To form. 1am Following BamHI digestion of DNA, BamHI (1.95 × 10 ⁶ mw) is lost and has a molar weight of about 1.45 × 10 ⁶ A new band appears below. Also connect BamHI containing Is not detectable but a new band with a water weight of 3.5 × 10 ⁶ , See below.

Taken together, the restriction enzyme profile showed that 1714 was determined in two copies of the terminal portions of 0-0.096mu and 0.81-0.83mu. The deletion was estimated to be 600-800 bp.

Southern blot analysis of 1714 DNA was performed to demonstrate loss of sequence in the R / L garment. 17 ⁺ digested with BamHI and a 1714 DNA was moved to a nitrocellulose membrane. 17 ⁺ DNA BamHI Fragments (+) were randomly initiated and hybridized to digested DNA. At ¹⁷⁺ tracks, the probe And Found hybridization with. In 1714 the probe Failed to hybridize to new New, with additional 'a' sequences , And Hybridized with. There is no hybridization of the furnace, but new Hybridized to. The joining of the size-ladder indicated that the deletion was about 800 bp. The results showed that 1714 was deleted in both copies of R _{L and} the deletion was BamHI. It clearly demonstrates that it is included.

(b) sequencing

BamHI of HSV-1 Strain 17 The linking fragment (s + g) is located between the nucleotide positions np 123459 and 129403 (McGeoch 1988-). BamHI from 1714 The fragment is deleted about 800 bp. This new BamHI in 1714 Cloned into the BamHI site of pGEM. In addition, restriction analysis indicated that the deletion strain was approximately 2.1 kb in size compared to the wild type 2.9 kb fragment and had deletions in the AluI fragment (125074-127066 np). This AluI fragment from 1714 was eluted from the agarose gel, redigested with SmaI and the resulting fragment was cloned into M13mp8.

The dideoxy sequencing of the SmaI fragment was 759 bp in length, so deletion was confirmed and located between nucleotide positions 125213 and 125972. From the remaining SmaI fragments sequenced, no other mutations were detected. The only correctly defined gene in R _L is IE1 at the 5 ′ end in IR _L 124108 np, ie 1105 bp downstream of the deletion. The IR _L / IR _S 'a' sequence in HSV-1 strain 17 starts at nucleotide position 125954. In 1714 exactly one complete 186 bp DRI element (AGCCCGGGCCCCCCGCGG) of sequence 'a' was removed.

Example 2

a) Neuropathy of deletion strain 1714 on Balb / c mice

We previously found that the deletion variant JH2604 of HSV-2 strain HG52 had no neuropathy in Balb / c mice with LD ₅₀ values of 10 ⁷ pfu / rat compared to 10 ² pfu / rat for wild type viruses. .

Sequencing of JH2604 demonstrated that the terminal portion of the genome long repeats (between 00-0.02mu and 0.81-0.83mu) provided neuropathy for strain HG52.

Since 1714 a deletion in the equivalent parts of the HSV-1 genome, By as compared to its parent 1702 and 17 ⁺ assess LD ₅₀ values in Balb / c mice was performed experiments to measure the nerve to forces of 1714.

25 μl aliquots of different doses of 17 ⁺ , 1702 and 1714 were inoculated into the left hemisphere of the 3 week old Balb / c mice. Death from encephalitis is recorded up to 21 days after inoculation and the results are shown in Table 1. Selected foci of accumulation of 17 ⁺ showed an LD ₅₀ value of <10 ^1.5 pfu / mouse. Mutant 1702, even tk negative (MacLean and Brown 1987a), provided higher LD ₅₀ values at the edge of 5 × 10 ² pfu / mouse. In 1714, animals were not killed by inoculation of 10 ⁶ pfu, but 3/4 of them died with 10 ⁷ pfu providing an LD ₅₀ value of 7 × 10 ⁶ pfu / mouse. Thus deletion variant 1714 was at least 2 × 10 ⁴ times less nerves and nerve disease onset St. St. higher at least 7 × 10 ⁵ times that of wild-type than the parent ⁺ 17 1702 biruseu. Single plaques were isolated from the brains of dead 1714 infected mice and the plaque DNA was digested with restriction enzymes. The RE profile was the same as that of 1714 and showed no wild-type contaminants. In the case of 17 ⁺ 72: the particle of 1: 1 and 58 in the case of 1714 pfu ratio is within equal and the value in the case of HSV-1 normal range.

(b) growth of 1714 in vivo

It has been found that HSV-2 (HG52) variant JH2604 is nontoxic; It did not replicate in mice and produced firm encephalitis (Taha 1990). In addition, samples of 17 ⁺ (10 ² pfu), 1702 (10 ² pfu), and 1714 (10 ⁵ pfu) were collected from 3 week old Balb / c mice to assess whether the absence of neuronal history of 1714 was due to replication failure in the mouse brain. Was inoculated into the left cerebral hemisphere. Two mice died at various times (per virus) after inoculation and their brains were detached and frozen at -70 ° C. Brain tissues were homogenized and the resulting suspension was sonicated and plaque titrated against BHK21 / C13 at 37 ° C. to assay for subsequent viruses.

The results showed that the virus grew exponentially between 12 hours and 6 days after infection in the parent strain 17, reaching a final titer of 8 × 10 ⁶ pfu / brain. Similarly in 1702, there was a virulent detectable 24 hours after inoculation and exponential growth reached an appropriate amount of 8 × 10 ⁴ pfu / brain by 6 days. In 1714 infected animals administered 10 ⁵ pfu and 2 × 10 ³ pfu, detection was possible immediately after inoculation. Replication could not be detected and the dosing virus lost until 3 days post inoculation was not an analytical virus (<10 pfu).

(c) growth of in vitro 1714

Variant 1714 grows to high titers (> 10 ⁹ pfu) by multicycle growth following low induction in BHK21 / C13 cells. Accumulations show equivalent titers when assayed at 31 ° C., 37 ° C., and 38.5 ° C. To measure its growth pattern, single cycle growth experiments were performed on BHK21 / C13 cells at 37 ° C. The results showed that 17 ⁺ and 1702 and 1714 strains are grown to equal the final yield to provide the same excellent. The normal single cycle growth pattern of 1714 shows no binding at any stage in its replicable cycle in BHK21 / C13 cells.

To determine if the virus is host-limited, a 24-hour counting experiment was performed in the range of cell lines infected with a pool of 5 pfu / cell. The cell lines used were BHK / C13 (hamster), BSC1 (monkey), Vero (monkey), MDCK (dog), HFL (human) and 3T6 (mouse). The 24-hour yield in BHK21 / C13 cells titrated at 37 ° C. is shown in Table 2 as the ratio of the yield of virus grown in a particular cell line compared to the yield in BHK21 / C13 cells. 17 ⁺ , 1702 and 1714 behave in a similar fashion in recent years; It can be seen that they grow equally well in BHK21 / C13, 3T6 and MDCK cells, better in Vero cells and less well in HFL and BSCI cells. Note that there was no replication defect in mouse 3T6 cells indicating that the defect of growth in vivo is not species specific.

Example 3

[Latent study]

Right posterior leggings of three week old Balb / c mice are inoculated with a series of 10-fold dilutions (4 mice / dose) of 17 ⁺ , 1702 and 1714 and monitored for signs of disease or death daily for two weeks. Six weeks after inoculation, surviving mice were dissected as outlined in the method (above) and nephrotic crystals were transferred separately to microtiter wells containing culture medium.

An aliquot of the culture medium was transferred to regulate BHK21 / C13 cells, so screening for the presence of infectious viruses was performed every second day after eating out. Cells were then incubated at 37 ° C. for 2 days and then stained and observed for the presence of virus plaque or cpe.

The results in Table 3 show that 20% of explanted ganglia are reactivated at doses of 17 ⁺ and 10 ⁴ and 10 ⁵ pfu. However, shortly after inoculation, 1 out of 10 ⁴ pfu infected animals and 3 out of 10 ⁵ pfu infected animals developed and had to die. All were predicted to die, so animals were not inoculated with 10 ⁶ pfu of 17 ⁺ . In 1702 infected animals, 5% of ganglia were reactivated at doses of 10 ⁴ and 10 ⁵ pfu and 17.5% reactivation at 10 ⁶ pfu doses. This is possibly clearly less efficient than 17 ⁺ due to the tk negative phenotype of the variant. In 1714 vaccinated animals, ganglia were not reactivated from 10 ⁴ pfu infected animals, only 1/40 (2.5%) reactivated from 10 ⁵ pfu infected mice and 2/40 (5% from 10 ⁶ pfu infected animals). ) Was reactivated. The virus was first activated 6 days after eating out and there was no significant difference in reactivation time between the three viruses. Virus reactivation was limited to lumbar ganglia in three groups of mice.

In view of the tk negative phenotype, variant 1714, although capable of incubating, is much less efficient than 1702 in establishing the incubation state and / or reactivating thereafter after eating out.

Example 4

[17 ⁺ Introduction of the 1714 deletion into wild type genome;

Since the 1714 deletion is not in the wild-type background, that is, four Xbal sites are deleted in the genome at 0.07, 0.29, 0.45 and 0.63mu and the virus is tk negative, it can be thought that these other mutations are at least partially non-toxic phenotypes. have. Parent strain 1702 containing the same Xbal-negative and tk-negative mutations appeared to be very dissimilar because it had a pathogenic phenotype that was essentially equivalent to 17 ⁺ . Nevertheless, we decided to introduce the deletions in 1714 into another entirely wild-type genome. A 17 ⁺ plasmid DNA of the clone 1714 the BamHI Co-transfect with 10-fold excess of. Single posterior plaques of the results were isolated and analyzed for their DNA profiles by the method of Lonsdale (1979). Viruses with a 1714 BamHI profile, designated 1716, were isolated, plaques were additionally purified three times and the virus accumulation was grown. To confirm that 1716 retained its wild-type background in terms of Xbal sites and tk activity, DNA of 1716 was digested with Xbal and tk assays were performed. It was found that 1716 had a normal wild-type Xbal profile with four sites in U _L while 1714 and 1702 did not digest with Xbal. 17 ^+, 1702 and 1714 as compared with the results of the tk assay for 1716 is provided in Table 4 indicate that 1716 is a composite 17 of tk ⁺ as effective. The neuropathic phenotype of 1716 was tested by IC inoculation of Balb / c mice. 17, whose LD ₅₀ value as compared to ⁺ and 1714 are presented in Table 5. Table 5 It was 7 × 10 ⁶ pfu / mouse of the LD ₅₀ value by non-neural onset adult On the other hand, the experiment in a 17 ⁺ a confirmation of providing care to a history about 1716 within the deletion sequence is strain 17, <10pfu / mice, the LD _It can be seen that it has a value of ₅₀ .

The results of the single cycle growth experiment by 1716 revealed that 1716 grew as effectively as wild type 17 ⁺ virus.

TABLE 1

* Dead numbers / inoculated numbers

ND = not inoculated

TABLE 2

Virus output over 24 hours at 37 ° C. was expressed as pfu / 5 × 10 ⁵ cells.

+ Ratio of virus yield in specific cell types compared to yield in BHK21 / C13 cells

TABLE 3

* Number of ganglia reactivated / number of conscious ganglia

+% Of ganglion reactivated

++ Four animals were infected / eating out 10 ganglia from each.

17 ⁺ in infected animals, 10 ⁴ pfu from one animal and 10 ⁵ pfu after 3 doses were put to death in the infected aroused the attack soon.

TABLE 4

TABLE 5

* Dead / inoculated

ND = no inoculation

Example 5

[Configuration of HSV-1 1716 gD1 ⁺ , gD2 ⁺ ]

HSV 2 strain HG52 (Mcgeoch Recombinant plasmids containing the Hind III 1 fragment of 1987a) were digested with restriction endonucleases Bst EII and Dra I and 3 Kb from the Dra I site of np 5893 to the Bst EII site of np 8893 were purified. This fragment contains a promoter, an open reading frame and a poly A signal of genes UL 6 (gD-2) and US 7 (gI-2) at the 3 'cavity end. The 5 'overhang of the Bst EII site was terminated flat using Klenow polymerase. The gD-2 containing fragments are integral to the integral membrane protein (Maclean C 1991), UL 43 (McGeoch 1988) into the Bam HI / ECoR1 91610/96751 np fragment of RSV1. The insertion site was the only Nsi I site np 94911 at the 5 ′ end of UL 43. The 5 'overhang of the Nsi I site was terminated flat using Klenow polymerase. All cloning techniques are as described by Maniatis Group in 1982.

Recombinant UL 43 gD2 HSV1 fragments were cotransfected with free HSV1 1716 variant DNA and the recombinant genome was isolated as described (Example 4 and Maclean). 1991). HSV recombinants containing gD2 were isolated. These viruses gD1 ⁺ gD2 ⁺ , ICP34.5 are distinguished as 1716.

Example 6

a) Composition of HSV-1 1716 U126 ts

ts 1201 (Preston 1983 cloned ECORI f fragment contains UL 26 with ts point mutation. This was recombined into 1716 to produce HSV-1716 UL 26 ts as previously described in Example 4.

HSV-1 1716 gD1 ⁺ gD2 ⁺ UL 26 ts.

b) HSV-1 1716 UL 26 ts and HSV-1 1716 gD1 ⁺ gD2 ⁺ from the above examples were recombined using standard methodology to provide HSV-1 1716 gD1 ⁺ , gD2 ⁺ , UL 26 ts viruses.

Example 7

Configuration of the ^{- 1716 gD1 +, gD2 + LAT} P - and ^{1716 gD1 + gD2 + UL 26 ts} LAP P

Fragments isolated from HSV-1 1704 1989, June Jo 1991) carries a 942 bp deletion in both copies of the LAT promoter. This fragment was cotransfected with 1716 gD1 ⁺ , gD2 ⁺ and 1716 gD1 ⁺ gD2 ⁺ UL 26 DNA and analyzed single plaques to yield 1716 gD1 ⁺ gD2 ⁺ LAP P ⁻ and 1716 gD1 ⁺ gD2 ⁺ UL 26 ts, LAP P ⁻ Provided.

HSV-1 strain 1714 and HSV strain 1716 were deposited on January 28, 1992 in the European Collection of Animal Cell Culture, Vaccine Research and Production Institute, Proton City Public Health Institute Services, Salisbury Wiltschatt SP4, OJ9, UK And the given accession numbers were V92012802 and V92012803, respectively.

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Claims (11)

HSV-1 lacking neuropathy, modified in the Bam H1 s region (0.81-0.83mu) of the internal repeat R _L and in the corresponding terminal R _L region (0-0.02mu) to the genome of the HSV-1 strain Variant of strain 17. The variant of claim 1, wherein the genome is modified by deletion in each region. The variant of claim 2, wherein at least 100 nucleotides are deleted from the genome of HSV-1 strain 17 in the corresponding region of the inner R _L and terminal R _L between nucleotides 125074 to 125972. The variant according to claim 2 or 3, wherein each of the deletions is 0.5-3 Kb in size. The variant of claim 4, wherein each of the strains is deleted from 0.7 to 2.5 Kb in size. HSV-1 1714. HSV-1 1716. 8. The variant according to any one of claims 1, 6 and 7, further modified by including a temperature sensitive mutation in the UL26 gene. 8. The variant according to claim 1, further modified by including a mutation which inactivates the LAT promoter. 9. Lightweight particles derived from variant 9. HSV-1 strain 17 introduces modifications in the Bam H1 s region (0.81-0.83mu) and in the terminal R _L (0-0.02mu) region of the internal repeat R _L to thereby lack the neuronal viability of the resulting strain. A method of producing a variant as claimed in claim 1, comprising a.